Various assays exist for measuring Antibody Secreting Cells (ASCs). One such assay is the antibody-secreting cell ELISpot which was first described in 1983 and is a method which enables detection of antibody secreting cells at the single cell level. The ELISpot can either be performed so that all antibody secreting cells or only the cells secreting antibodies specific for a particular antigen are detected. By using specific reagents, one can also restrict the detection to a certain immunoglobulin (Ig) isotype (for example, IgG, IgA or IgE) or IgG subclass (for example, IgG1, IgG2 or IgG3).
For the antigen-specific ASC ELISpot, the antigens to be tested are coated onto the membrane of ELISpot plates to which the ASCs are added and incubated for various time spans (from a few hours to several days). During this time antibodies secreted from individual cells may bind to the coated antigen at the site of the secreting cell. After removing the cells, the specifically and locally bound antibodies are visualized by the addition of labelled (for example with an enzyme or biotin) anti-Ig antibodies. If using biotin, an additional step with enzyme-labeled Streptavidin or avidin is required before finally developing the plates by the addition of a precipitating substrate. Each spot observed on the ELISpot plate corresponds to one cell producing the correct antibody and the spots can be counted either manually under a microscope or in an ELISpot reader.
The ELISpot assay may be used to detect all antibody-secreting cells regardless of their specificity by coating the ELISpot plates with anti-Ig antibodies and then visualizing the bound antibodies using labelled anti-Ig antibodies in the same way as for the antigen-specific ASC ELISpot. When the total Ig ASC ELISpot is carried out in parallel to the antigen-specific ASC cell ELISpot as a positive control, the frequency of specific ASCs can be calculated from the number of spots observed in each assay.
A negative control with uncoated wells may also be used. No spots should be observed in these wells. However, the negative control may not always be negative. Spots occurring in negative control wells may represent non-specifically binding “sticky” antibodies or antibodies reactive to proteins, such as BSA or serum used for blocking the ELISpot plates.
The conventional antigen-specific ASC ELISpot assay described above has several other disadvantages. For example, to obtain good quality spots, a large amount of the antigen has to be used to coat the plates and the scarcity and cost of many antigens may be limiting in many situations. Further, due to the uncontrollability of binding and immobilization of antigen to the ELISpot plate, relevant antigen epitopes can become unavailable for antibody binding, either through “masking” (i.e. the epitope is involved in the binding to the plate and thereby made inaccessible) or denaturing of the conformational structure of the protein when immobilized to the plate. In addition, some proteins (particularly smaller peptides) as well as other substances (e.g. carbohydrates and lipids) may not bind efficiently to the ELISpot plate.
The antigen-specific ASC ELISpot can also only readily be carried out to test one antigen in each well of the ELISpot plate as it is not possible to distinguish and separately count the cells producing antibodies to the different antigens.